Amadori reaction compounds and products, process for their manufacture, and their use as cytokine inducers

ABSTRACT

Novel Amadori reaction compounds have the formula R 1  --NH--R 2 , wherein R 1  comprises the D-form of a 1-amino-1-deoxy-2-ketose radical derived from a sugar radical selected from the group of glucose, xylose, galactose, rhamnose, fructose, mannose, 6-deoxyglucose, glucosamine and galactosamine, and R 2  comprises the L-form of an aminoacid or peptide radical selected from the group of serine, glycine, proline, histidine, arginine, alanine, aspartic acid, glutamic acid, phenylalanine, treonine, cysteine, cystine, glutamine, asparagine, methionine, tyrosine, hydroxyproline, tryptophane, valine isoleucine, lysine and leucine. Compounds and combinations of compounds having the general formula R 1  &#39;--NH--R 2  &#39;, wherein R 1  &#39; comprises a 1-amino-1-deoxy-2-ketose radical derived from the group of simple sugars, oligo- and polysaccharides, and R 2  &#39; comprises an aminoacid or a peptide radical, are used to produce pharmaceutical preparations which in contact with human leukocytes produce interferon and other cytokines.

This application was filed under 35 USC 371 as a national stage ofInternational case PCT/EP93/00327 filed Feb. 11, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention related to novel Amadori reaction compounds andproducts, to the production thereof and to a new use of these compoundsand products, having at least partly entered an Amadori rearrangement asper the following reaction scheme and/or a Maillard reaction: ##STR1##Amadori reaction products are known; they are reaction products, forexample, of an aminoacid or a peptide with a sugar, oligo- orpolysaccharide having entered an Amadori rearrangement (J. Biol. Soc.215 (1955), Henri Borsock et al.). Thus, in DE-C-3914354, awater-soluble glycoprotein of an aminoacid and a sugar is describedwhich is isolated from an extract of Avena sativa seeds. Further,EP-A-406087 describes water-soluble polysaccharide-glycopeptidecomplexes which are derived from the cell wall of Gram positivebacterium, and J. Biol. Chem. 1985, 260/9 states that NMR-spectroscopyhas been used to characterize Amadori reaction products formed byreaction of glucose with free amino groups of protein.

2. Description of the Related Art

The invention now describes specific novel Amadori rearrangementcompounds.

These compounds reduce potassium ferricyanide, a test reaction forbiological active substances formed in a reaction of sugar andaminoacid.

The invention further relates to a novel use of such compounds andsimultaneously a novel use of Amadori reaction products of sugars andaminoacids in general. In the past, nobody has tested the various stepsof purification of the extract of an Amadori reaction product (in orderto get ride of ballast substances and impurities) for biologicalactivity.

Immunostimulating drugs are indeed known from natural sources such asmistletoe extracts, peat extracts etc. with the drawback of expensivetreatment of large quantities of raw material to obtain a few grams ofactive substance. Uncontrolable impurities might lead to toxicity andside effects and therefore, further, to problems with administrationduring practical use due to the complex nature and the hardlyreproducible composition.

Comparable products or product mixes from artificial sources, such asinterferon or other genetic engineering methods are even more expensiveto make furthermore, the molecules of human interferon are very oftentoo big to penetrate the human cell wall so that only a fraction of theadministered dose is effectively becoming active. Also, geneticengineering products usually have side effects and some are even toxicfurthermore, some of them act effectively on one day and not on the nextday, for reasons unknown so far.

Surprisingly, it has now been found that nearly any simpleamino-acid/sugar complex after having at least partly entered an Amadorirearrangement does not show any of the above-mentioned disadvantages buton the contrary has a surprisingly high immuno-logical activity. Theycan therefore be used in pharmaceutical formulations and in cosmetics.These small molecules easily penetrate the cell wall and virtually actas a nutrient. They induce the formation of natural interferon and othercytokines, including tumor necrosis factor. Even three days after theadministration, they still show this stimulating effect on biologicalactivity. This effect increases with increasing completion of theAmadori rearrangement and decreases again with increasing decompositionof the complex.

Instead of simple sugars also--preferably low molecular weight,especially of less than 1000 daltons--polysaccharides may be used, forinstance dextran, which react similarly. Polysaccharides show biologicalactivity and may retain some of this activity after they becomeoligosaccharides.

Very little has been known heretofore about the biological activity ofthese compounds. It was now found that combinations of these substancesin contact with human leukocytes produce interferon and other cytokines.This is called polyclonal activation of the cells.

It is possible to rest the substances produced under the influence ofthese compounds and to determine the biological activities ininternational units relevant to specific cytokines. These compounds areof especially high biological activity within a range of pure substanceconcentrations from 1-100 μg/ml. Within the molecule, the specificnature of the aminoacid is more important than the nature of the sugarpart of the molecule.

Reaction products of L-aspartic acid with glucose or galactose--afterhaving gone through the Amadori rearrangement--when contacted with humanleukocytes and incubated in tissue culture mediate at 37° C. for 20hours in an atmosphere of 5% CO₂ will produce from 30-1000 antiviralunits of interferon. The interferon is measured in a bioassay usinghuman cancer cells. Under the influence of these compounds, tumornecrosis compounds may also be produced.

The products which allow such an unexpected use have the general formula

    R.sub.1 '--NH--R.sub.2 '

wherein

R₁ ' represents a 1-amino-1-deoxy-2-ketose radical derived from thegroup of simple sugars, oligo- and--preferably low molecular weight,especially of less than 1000 daltons--polysaccharides, and

R₂ ' represents an aminoacid or a --preferably low molecular weight,especially of less than 1000 daltons--peptide radical.

Thus the group of biologically active compounds may cover either thespecific Amadori rearrangement compounds described above or theN-substituted derivatives of a number of different aminoacid compoundsand one simple sugar, oligo- or --preferably low molecular weight,especially of less than 100 daltons--polysaccharide, or N-substitutedderivatives of one aminoacid compound and a number of simple sugars,oligo- and/or such polysaccharides, or else any combination of suchderivatives, every single one of them having sufficient biologicalactivity.

Preferably, R₁ ' in the above formula may be a radical selected from theD-form of simple sugars, especially (but not exclusively) from theD-form of glucose, xylose, galactose, rhamnose, fructose, mannose,6-deoxyglucose, glucosamine and galactosamine; R₂ ' may be a radicalselected from the L-form of aminoacid compounds such as serine, glycine,histidine, arginine, glutamine, asparagine, alanine, aspartic acid,glutamic acid, phenylalanine, treonine, cysteine, cystine, methionine,hydroxyproline, tryptophane, proline, tyrosine, valine, isoleucine,leucine and lysine or else peptides of these aminoacids in anycombination.

The invention further relates to a process for obtaining the abovementioned compounds and products and whereby an intermediate is formedof the formula

    R'--NH--R"←STAT

wherein

R' is a 1-deoxy-2-Ketase radical in a straight carbon chain or anyO-bridged form of a simple sugar or an oligo- or polysaccharide, and

R" represents an aminoacid or a peptide radical, said intermediate beingat least partly subjected to an Amadori rearrangement and/or to theMaillard reaction by continued heating of the reactionmixture--preferably under pressure--and simultaneously or subsequentlyremoving the solvents.

In some cases, especially when an aminoacid having two carboxyl groupsis used, it is advantageous to add to the process a buffer salt, such assodium bicarbonate, preferably in a molar ratio of 1:1.

It was further found that the amadori rearrangement products arerelatively susceptible to decomposition, and that decomposition productshave the nature of dark brown and tar-like, unidentified compoundshaving lost their biological activity. Therefore it is preferred to stopthe Amadori rearrangement reaction at a stage at which the reactionmixture becomes light orange-brown in color.

It is interesting to note that the intermediate reaction products,formed when the originally opaque solution of the aminoacid becomesclear (before the Amadori rearrangement), are easily hydrolyzed, i.e.the reaction is reversible. With increasing rearrangement, thereversibility diminishes, i.e. the products become more stable, and thecolor gradually changes from light yellow to light orange, and thenfinally to orange-brown when the Amadori rearrangement seems to becomplete. Samples taken during such rearrangement reaction and tested(according to various procedures described later) proved that thebiological activity increases with the Amadori reaction progressing, anddecreases when further heating results in decomposition, for which acolor change to dark brown is a sign. Immediate reduction offerricyanide and the resulting color change will occur if the reactionmixture contains other keto groups and/or sulfur containing aminoacidssuch as cysteine; otherwise it will occur within 3 to 5 min, which is agood check for the degree of Amadori rearrangement developed. Unreactedsugars would show the color change after half an hour or several hoursonly.

Isolation of the pure Amadori rearrangement products is carried outaccording to known methods based on binding the mixture on a strongcation-exchanger (such as Amberlite® or Dowex®, successively elutingwith ammonia water, evaporating a chosen defined fraction of the eluateunder reduced pressure and crystallising the pure compound fromanhydrous methanol (J. E. Hodge and B. E. Fisher, Methods inCarbohydrate Chemistry, Vol. II, Reactions of Carbohydrates, AcademicPress, N.Y., London, 1963, page 105-106; or Borsook et al. as quotedearlier; or J. Dubourg and P. Devilliers).

In the process according to the present invention, all theabove-mentioned preferences regarding the kind of radicals derived fromsimple sugar and aminoacid compounds remain unchanged. Additionally, apreferred mixture of sugar substrates comprises the D-forms of glucose,xylose, galactose, rhamnose and fructose in a weight ratio of about20:10:4:1:1, while the preferred mixture of aminoacid substratescomprises the L-forms of serine, glycine, histidine, arginine, alanine,proline, tyrosine, valine, leucine, isoleucine and lysine in a weightratio of 20.5:35.8:35.8:132: 180:360:216:160:72:68:780.

As already mentioned, the Amadori rearrangement products are able toreduce potassium ferricyanide, such a chemical test reaction provides abasis for quick determination of the biological activity of thecomposition formed in a reaction of sugar and aminoacid.

It has been found that Amadori reaction products are especially activeif a mixture of simple sugars of the same composition and in the sameweight ratio as occurring in natural peat extracts are reacted with amixture of aminoacid compounds of the same composition and in the sameweight ratio as occurring in natural peat extracts in the presence of anaqueous solvent, preferably adding a lower alcohol--and optionallyinorganic trace elements occurring in such natural peat extracts--atelevated temperature (and optionally under pressure), and subsequentlyprolonging the heating in order to cause an Amadori rearrangement of theobtained products, simultaneously or subsequently expelling thesolvents, stopping the rearrangement reaction at the point when thereaction mixture becomes light orange-brown in color, drying theproducts thus obtained and purifying the same by means of columnchromatography and collecting the fractions that cause maximum reductionof potassium ferricyanide.

In one embodiment, the instant pharmaceutical formulations contain as anactive ingredient at least one reaction product of the formula R₁'--NH--R₂ ' or a specific compound of the formula R₁ --NH--R₂ togetherwith a pharmaceutically acceptable carrier and/or an adjuvant and/oroptionally a lubricant in a weight ratio of active ingredient to theremaining components of between 1:1 and 1:100, preferably 1:8 to 1:20and most preferably about 1:9.

Another advantageous pharmaceutical formulation contains--in addition tothe active ingredient--lactose and a lubricant; the weight ratio oflactose to the lubricant being between 20:1 and 100:1, preferably 50:1.

These pharmaceutical preparations are used to treat and/or preventhematological and/or immunological diseases, and/or stimulate theimmunosystem of humans and/or mammals by the induction of cytokineformation.

Another use for the active ingredients is in cosmetic preparations. Theactive ingredient is present in such preparations in amounts of 0.01-10%by weight, preferably 0.01-1% by weight and especially in amounts of0.05-0.1%. These cosmetic preparations contain--besides the activeingredient--usual carriers, adjuvants, enriching components and/orfragrants.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be further explained and demonstrated in thefollowing examples, which do not limit in any respect the scope of thepresent invention.

EXAMPLE 1

A 25 ml flask of a rotary evaporator placed in a heated water bath wascharged with:

1.47 g (0.01M) L-glutamic acid

0.84 g (0.01M) NaHCO₃

0.91 g D-glucose

0.91 g glactose and

3.00 ml of redistilled water

The mixture was heated up to 80° C. whereby--preferably under reducedpressure--50% of the water was evaporated and then--under atmosphericpressure--heated to a temperature of 80°-85° C., at which it was keptfor 120 min until it became orange in color. The syrup-like concentratedaqueous solution was then evaporated to dryness under reduced pressure.The orange-red solid reaction product thus obtained was marked with thesymbol D-10 and saved for biological tests.

EXAMPLE 2

A 25 ml flask of a rotary evaporator placed in a heated water bath wascharged with:

1.33 g (0.01M) L-aspartic acid

0.84 g (0.01M) NaHCO₃

0.91 g of D-glucose

0.91 g of galactose and

3.00 ml of redistilled water

The mixture was heated up to 80° C. with stirring (by means ofrotation), concentrated under pressure, whereby a total volume of 1.5 mlwater was vaporized, and then treated under atmospheric pressure at atemperature of 80°-85° C. until it became light orange in color; thistook place within approx. 60 min.

The reaction mixture was a syrup-like concentrated aqueous solution andwas dried under reduced pressure. The resulting reaction product was adry, yellow-orange powder. It was marked with the symbol D-11 and savedfor biological tests.

EXAMPLE 3

A 25 ml flask of a rotary evaporator placed in a heated water bath wecharged with:

1.05 g (0.01M) L-serine

0.91 g D-glucose

0.91 g galactose and

2.50 ml redistilled water

The mixture was heated to 85°-92° C. with stirring (by means ofrotation). After 100 min, the solution became orange in color. Thepressure was reduced and the mixture was evaporated to dryness. Thereaction product on the walls of the flask formed an orange transparentlayer. The reaction product was scraped off and powdered. It was markedwith the symbol D-12 and saved for biological tests.

EXAMPLE 4

A 25 ml flask of a rotary evaporator placed in a heated waterbath wascharged with:

0.66 g (0.005M) of D-aspartic acid

0.42 g (0.005M) of NaHCO₃

0.45 g of D-glucose

0.45 g of galactose and

3.00 ml of redistilled water

The mixture was heated up to 80° C., evaporated--under pressure--wherebya volume of 1.5 ml water was vaporized, and then treated--underatmospheric pressure--at a temperature of 85° C. After 60 min. heatingthe mixture became orange in color; the pressure was reduced and theresulting syrup-like concentrated aqueous solution evaporated todryness. Before the residue became definitely dry, two times 10 mlwater-free ethanol were introduced into the flask and evaporated inorder to eliminate residual moisture. The dry reaction product thusobtained was powdered, marked with the symbol D-13 and saved forbiological tests.

EXAMPLE 5

In order to obtain--in a synthetic way--an equivalent of thebiologically active fraction of a certain natural peat extract, theflask of the rotary evaporator placed in the heated water bath wascharged with:

20.5 mg L-serine

35.8 mg L-glycine

35.8 mg L-histidine

132.0 ; mg L-arginine

180.0 mg L-alanine

360.0 mg L-proline

216.0 mg L-tyrosine

160.0 mg L-valine

68.0 mg L-isoleucine

72.0 mg L-leucine

780.0 mg L-lysine

2000.0 mg D-glucose

1000.0 mg D-xylose

400.0 mg D-galactose

100.0 mg D-rhamnose

100.0 mg D-fructose

6.0 ml redistilled water

The mixture was stirred by means of rotation and heated under pressurefor 45 min at a temperature rising from 75° C. to 86° C. During thatperiod, approx. 3 ml of water were evaporated and the substrates weretotally dissolved. The mixture was then treated for 30 min underatmospheric pressure at a temperature of 85°-86° C. for an Amadorirearrangement. During that period the solution quickly became red-brownin color. The pressure was reduced, and heating at 84° C. was continued,thus simultaneously evaporating the solvents. At the end of evaporation,two times 15 ml water-free ethanol were introduced and the reactionmixture was brought to dryness. The flask with the dried reactionproduct was placed in a desiccator over calcium chloride for 18 hours;then the reaction product was powdered. Approx. 4.5 g of a powderedproduct were obtained and marked with the symbol EK₂ -S.

A portion of 4 g of this reaction product was dissolved in 20 ml ofdistilled water and placed on a chromatographic column of 25 mm×330 mmsize, filled with a sorbent Amerlite ® XAD-2 analytical grade. Thecolumn was eluted with 0.4 ml/min distilled water. Fractions of 10 mlvolume were collected to a total volume of 450 ml. The content of thefractions was monitored chromatographically. Fractions of consecutivenumbers 11-13 were combined and evaporated under reduced pressure. Thesefractions were characterised by a high content of Amadori rearrangementproducts (confirmed with the potassium ferricyanide reduction test). Theproduct was saved for biological tests under the symbol of EK₂ -S-11.

Biological tests for determining the biological activity were carriedout with immunised Balb/C mice of both sexes, at the age of 8-10 weeks.Immunization of mice is achieved by peritoneal administration of 0.2 mlof a 10% suspension of sheep erythrocytes (SRBC), i.e. of 6×10⁸ cells.The erythrocytes are fixed in a sterile Alsever's solution of thefollowing composition:

    ______________________________________                                        glucose                2.05 g                                                 sodium citrate         0.8 g                                                  sodium chloride        0.42 g                                                 citric acid            0.055 g                                                redistilled water to   100 ml                                                 ______________________________________                                    

Into such Alsever's solution, a sheep blood cell aseptic sample isintroduced in a ratio of 1:1 and the mixture is kept for at least 3 daysat +4° C. The thus stabilised erythrocytes are then sampled asepticallyand introduced into a phosphate buffered salt solution (PBS) in order towash them out. Erythrocytes are rinsed with PBS twice, and arecentrifugated for 10 min at 2000 rpm. The washed out cells are used inthe form of a 10% suspension in PBS. Such a suspension is used for theimmunization of Balb/C mice.

The reaction product to be tested was administered intraperitoneally(i.p.) or orally (p.o.) four times at chosen doses, the firstadministration taking place 2 hours before immunization of the mousewith SRBC, while the remaining three dosages were administered afterimmunization at 24 h intervals.

Each test group of animals was treated with different doses of thetested reaction product: 10 mg/kg, 1 mg/kg, 0.1 mg/kg and 0.01 mg/kg. Acontrol group of animals was also immunized with SRBC, but instead ofthe substance to be tested, 0.2 ml of PBS were administered at the sametime intervals.

Each group of animals, control and tested groups, in all experimentsconsisted of 8-12 mice.

On the fourth or (in case of determination of antibodies type 7S) tenthday after immunization, mice were slightly anesthetized with ether andexsanguinated by eyeball extirpation. The blood was collected into testtubes. Next, the spinal cord was broken and spleen removed. The bloodwas used for obtaining the serum needed for determination ofhemagglutinating antibodies of the 19S+7S and 7S types, while spleenswere used to prepare the cells useful for determination of thepercentage of cells able to form E-rosettes and of hemolytic activity.For such uses, mouse spleens were comminuted. The splenocyte cellsobtained were suspended in approx. 2 ml of Hanks' medium at +4° C.,layered on the Ficoll-Uropolin gradient of a density of 1.077, and thencentrifugated for 15 min at 3000 rpm at +4° C. After separation from theinterphase, the lymphocyte buffy coat was placed in the Hanks' medium at+4° C. and rinsed twice with centrifugation for 7-10 min each time at1800 rpm. The splenocytes were then suspended in 1 ml of Hanks' mediumat such a ratio that it contained 1×10⁶ cells.

For each test, the percentage of dead cells is determined by mixing adrop of a tested suspension of splenocytes with a drop of ex temporeprepared dyestuff solution containing 4 parts of a 0.2% trypan bluesolution and 1 part of a 4.25% NaCl solution. Under the microscope, thepercentage of dead splenocytes is determined for each 100 cells. Deadcells are navy blue, while the bright cells are live cells. The presenceof more than 10% of dead cells is critical; such a sample has to beeliminated from further use.

All steps carried out with the cells to be tested are performed in asterile, siliconized laboratory glass apparatus placed in an ice bath.

EXAMPLE 6

In the first test, an effect of the tested reaction products on thenumber of cells producing hemolytic antibodies (PFC-IgM) was determined.The test was carried out as follows: To 0.5 ml of an 0.5% agarosesolution placed in a test tube and kept in a heated water bath at 45°C., 0.1 ml of a 10% suspension of SRBC (prepared as described above)were admixed. Then 0.1 ml of a splenocyte suspension having a density of1×10⁶ cells/ml was added, the mixture stirred rapidly and immediatelypoured out on slides previously covered with agarose. The slides areincubated at 37° C. for two hours. Next, the tested samples are coveredwith guinea pig complement diluted at a ratio of 1:20 for a further 2 h.After the incubation of the tested samples with the complement, thenumber of plaque forming cells (PFC) was counted and recalculated for1×10⁶ splenocytes. Each test was performed twice.

The strongest amplification of the response to SRBC expressed in termsof increase of the number of splenocytes producing hemolysines IgM (PFC)was observed after administration of the D-11 substance at a dose of 0.1mg/kg. The amplification was 119%. When the daily dose was increased tentimes up to 1 mg/kg, amplification of the response was lowered to 53%.

Reaction product D-12 showed the strongest activity--an increase of58%--at a dose of 1 mg/kg.

Reaction product EK₂ -S-11 in this test showed the strongest activity ata dose of 0.1 mg/kg (increase of 65%). At a dose ten times higher, i.e.1 mg/kg, the increase was slightly lower, i.e. 52%.

Reaction product D-13 tested at a dose of 1 mg/kg caused an increase of40%. When the dose was increased to 10 mg/kg, i.e. ten times, theresponse was only a 14% increase.

EXAMPLE 7

An active hemagglutination test was also carried out, determining thelevel of the anti-SRBC type 19S-7S and 7S the level. In order todetermine the 19S-IgM type antibodies level, mouse serum was prepared onthe fourth day after immunization with SRBC, while for the determinationof 7S-IgG type antibodies level such preparation took place on the tenthday after mice immunization with SRBC, which is related to the day ofmaximum count of antibodies of a given type in mice immunized with SRBC.

A. Determination of 19S+7S antibodies count.

A sample of blood was centrifuged for 30 min at 3500 rpm. From eachsample thus prepared, serum was collected and placed in a heated waterbath at a temperature of 56° C. for 30 min in order to deactivate thecomplement. Next, a number of solutions at several different dilutionsof each tested serum was prepared (from 1:1 to 1:4096) employing amicrotitrator and U-shaped microplates having a volume of 250 μl each.The diluted sera were incubated for 1 hour at room temperature. A dropof a 1% suspension of SRBC in PBS (prepared as described above) wasadded to each serum; the mixtures were incubated for a further 2 h at atemperature of 37° C. and then stored at a temperature of +4° C. Theresults were taken next day. The maximum dilution at whichhemagglutination is still caused was considered the anti-body. count. Aring at the bottom of the plate is a sign that hemagglutination occurs.A button-like formation at the bottom of the plate is considered as anegative result--no hemagglutination.

For statistical analysis of the results, the increase of dilution ofserum in a tested substance was compared to the one in a control group.

Reaction product D-11 at a dose of 1 mg/kg increased the IgM count 2.57times. At a ten times higher dose, the stimulation effect in comparisonto the control group increased by 3.5 times.

Reaction product D-12 in this test showed a weaker activity. At a doseof 0.1 mg/kg, it increased the IgM count 2 times, and at a dose of 1mg/kg 1.4 times.

Reaction product EK₂ -S-11 showed the strongest activity in this test.At a dose of 0.0 mg/kg, it increased the IgM count 4.3 times, and at adose of 1 mg/kg 3.6 times.

Reaction product D-13 at a dose of 1 mg/kg increased the IgM count 3times, and at a ten times higher dose 1.5 times.

B. Determination of 75 antibody count

The tested inactivated sera were combined--at a ratio of 1:1--with a 0.1M solution of 2-mercaptoethanol and the mixtures incubated for 30 min ata temperature of 37° C. 2-Mercaptoethanol destroys immunoglobulins ofthe 19S-(IgM) type, while immunoglobulins of the 7S-(IgG) type are notsusceptible to the action of 2-mercaptoethanol.

After 30 min of incubation, the reduction reaction was stopped by meansof cooling down to a temperature of +4° C. for 15 min. Next, a number ofdilutions was prepared in a similar manner as described above withrespect to determining the 19S-antibody count and combined with a 1%suspension of SRBC; after 2 hours of incubation at 37° C., the sampleswere stored at a temperature of +4° C. The results were evaluated on thefollowing day, according to the criteria of determining thehemagglutination count described above. Simultaneously, a control testwas carried out with a combination of a 1% suspension of SRBC with PBSin a ratio of 1:1.

When the substance D-11 was tested as described above, at a dose of 1mg/kg it increased the production of antibodies IgG 3.16 times. At adose of 10 mg/kg, the increase was 2.2 times.

Reaction product D-12 tested at a dose of 0.1 mg/kg and 1 mg/kgrespectively stimulated the production of antibodies IgG 1.3 times, andat a dose of 10 mg/kg 1.5 times.

Reaction product EK₂ -S-11 at a dose of 0.1 mg/kg stimulates theproduction of IgG 1.9 times, and at a dose of 1 mg/kg 2.89 times (incomparison with the control).

The results of tests A and B obtained for each production lot or foreach fraction of the biologically active reaction products synthesizedaccording to the invention and giving the above-mentioned immunologicalresponse were subjected to statistical analysis by the T-student method,α=0.05. Results obtained for each dose tested were compared with aparallel control test and showed an increase of biological activity.

EXAMPLE 8

The group of biologically active reaction products obtained according toExamples 1 to 5 have also been submitted to the test in which thepercentage of splenocytes forming E-rosettes was determined.

250 μl of a 1% suspension of SRBC and 250 μl of cells to be tested at aconcentration of 1×10⁶ cells/ml were added to 550 μl of Hanks' medium.Each such samples was incubated in a heated water bath equipped with ashaker for 15 min at a temperature of 37° C. Then, it was stored at atemperature of +4° C. for a further 20 h. The percentage of splenocytesforming E-rosettes with SRBC was determined after the suspension wascolored with 1 to 3 drops of crystal violet.

Each sample was subjected to determination of the percentage ofsplenocytes three times, counting at each instance 400 splenocytes. Asplenocyte surrounded with at least 3 erythrocytes was considered anE-rosette.

For statistical evaluation, the percentage increase of the number ofsplenocytes with E-rosettes was compared between the substances to betested and a control group.

In this test, the strongest stimulating effect was shown by the reactionproducts D-11 (63%) and EK₂ -S-11 (70%) at a dose of 1 mg/kg. At a doseten times smaller, i.e. 0.1 mg/kg, the values decreased to 45% and 57%respectively.

Reaction product D-12 at a dose of 1 mg/kg caused an increase of theability to form E-rosettes of 22% in comparison to the control group.The respective value for a dose ten times smaller, i.e. of 0.1 mg/kg,was 29%.

Reaction product D-13 shows the maximum effect at a dose of 1 mg/kg(58%), while at higher doses the effect is slightly smaller.

Biological activity of synthesized compounds was evaluated according tothe following tests:

1. Test for determination of the percentage of splenocytes formingE-rossettes, carried out according to Bach and Dardenne (Cell. Immunol.3, 1-16, 1972)

2 Test for determination of the number of cells producing hemolyticantibodies of an IgM type, carried out according to the Jerne method,modified by Mishell and Dutton (J. Exp. Med. 126, 423-442, 1967) and

3. Test for determination of a hemagglutination 19S-7S and 7S antibodycount, carried out according to Adler's active hemagglutination methods(J. Immunol. 95, 26-38, 39-47, 1965) with the use of microplates (J.Immunopharmacol. 4, 43-52, 1982).

EXAMPLE 9

A rotary evaporator flask placed in a heated water bath was chargedwith:

1.33 g (0.01M) L-aspartic acid

0.84 g (0.01M) NahCO₃

10.00 g hydrolyzed dextrane of an average molecular weight of 3000daltons

10.00 ml redistilled water.

The mixture was heated under pressure at a temperature of 70° C. untilthe solid substances were completely dissolved, expelling by means ofdistillation during that period approx. 3 ml of water (heating time wasapprox. 30 min). The flask with the solution was loosely covered, placedin a steam sterilizer and heated under pressure at a temperature of 121°C. for 40 min. After cooling down, the resulting yellow-orange solutionwas diluted with 15 ml of water, clarified by means of centrifugationand spray dried by air having an inlet temperature of -160° C. and anoutlet temperature of +85° C. 10.5 g of a light beige reaction productresulted that was easily soluble in water.

The presence of Amadori rearrangement products in this reaction productwas confirmed by a test by the potassium ferricyanide method describedby Borsook, Abrams and Lowy, J. Biol. Chem 215, (1955), 111-124 and bychromatographic methods.

Biological tests as described in the preceding Examples confirmed animmunotropic activity of the above product similar to the one exhibitedby preparations obtained with simple sugars.

EXAMPLE 10

A conical flask was charged with:

5.0 g hydrolyzed dextrane of an average molecular weight of approx. 5000daltons

1.1 g L-proline

4.0 ml redistilled water.

The content was dissolved by means of stirring. The uniform mixture thusobtained was placed in a steam sterilizer and heated under pressure at atemperature of 110° C. for 40 min. The resulting transparent orangesolution was diluted with 20 ml of redistilled water and clarified bymeans of centrifugation. The clear solution was spray dried.

5.3 g of a reaction product easily soluble in water was obtained.Immunotropic activity was similar to the one observed in otherexperiments according to the preceding examples.

EXAMPLE 11

Conventional methods test the biological activity of the compounds inmice, but not in humans. For this reason, new bio-assays have beenintroduced, which measure the amounts and activity of cytokines releasedfrom the human peripheral blood leukocytes (PBL) treated with thereaction products according to the Examples 1 to 5, 9 and 10. Thecytokines are the hormone-like proteins that play an important role inpractically all of the immunological reactions, as well as in theregulatory processes responsible for the maintenance of homeostasis.

Cytotoxicity assays.

Cytotoxicity of the reaction products was determined in human lungadenocarcinoma cell line A549 (included in the American Type CultureCollection--ATCC CCL 185). The cell monolayers were trypsinized,suspensions of 2×10⁵ cells/ml in Dulbecco's-modified Eagle's minimumessential medium (DMEM) plus 10% calf serum (CS) were mixed with variousdoses of the drugs, seeded in the plastic microplates, and incubated for48 h at 37° C. CD₅₀ was the minimal concentration of the compound whichcaused approximately 50% destruction of the cell culture as measured bystaining with 0.015% solution of neutral red in DMEM.

Cytokine induction.

Buffy coats from healthy blood donors were obtained from the regionaltransfusion center. The erythrocytes were lysed by NH₄ Cl treatmentaccording to Cantell et al. (Cantell, K., Hirvonen, S., Kauppinen, H.L.: Production and Partial Purification of Human Immune Interferon.Meth. Enzymol., 119, 54, 1986). The leukocytes from a single donorcontaining approximately 8×10⁶ leukocytes/ml in RPMI 1640 mediumsupplemented with 10% fetal calf serum (FCS), L-glutamine, andantibiotics were used. All lots of FCS were pretested. Onlynon-mitogenic FCS for PBL cultures was used. The cytokine inducers wereadded to 1 ml volumes of the cultures. The reference cytokine inducerswere phytohemagglutinin (PHA) (Pharmacia Fine Chemicals, Sweden) and LPSfrom E. coli 0111:B4 (Difco Laboratories). The induced cultures of PBLwere incubated in an atmosphere of 5% Co₂ in air at 37° C. for 20 h andcentrifuged. Supernatants were stored at 4° C. and assayed for TNF andIFN activity within one week.

Interferon (IFN) assay.

The confluent monolayer of A549 cells was prepared in the microplates inDMEM with 10% CS, L-glutamine, and antibiotics (100 units/ml penicillinand 100 μg/ml streptomycin). IFN samples diluted in plates were added tothe cell monolayer and incubated at 37° C. for 20 h in 5% CO₂ in air.The cells were then washed and challenged with encephalomyocarditisvirus (EMCV). The titer of IFN was defined as the dilution of IFN samplethat reduced the virus cytopathogenic effect by 50% after 48 h ofincubation. The MTT (3-4,5-dimethylthiazol-2-yl!-2,5-diphenyltetranolium bromide) method(Hansen, M. B., Nielsen, S. E., and Berg, K.: Re-examination and FurtherDevelopment of a Precise and Rapid Dye Method for Measuring Cell Growth/Cell Kill. J. Immuno. Meth., 1989, 119, 203-210) to measure the cellkill in the ELISA scanner was also used. Laboratory standards of IFNswere included in all assays: Recombinant human IFN-γ (Genentech Inc.,USA, specific activity 2×10⁸ units/mg), the natural human leukocyteIFA-α (3×10⁶ IU/ml) and IFN-γ (2×10⁶ IU/ml) obtained from Dr. K.Cantell, Helsinki, Finland.

Tumor Necrosis Factor (TNF) assay.

The cytotoxic activity of TNF was measured in L929 cells according toFlick and Gifford (Flick, D. A., Gifford, G. E.: Comparison of in VitroCell Cytoxic Assays for Tumor Necrosis Factor. J. Immunol. Meth., 68,167, 1984).

The sample and actinomycin D solution were added to monolayer culturesof the cells. After incubation at 37° C. for 20 h, the cultures werestained with crystal violet and toxic effects were determined. Theamount causing approximately 50% destruction of the cell cultures wasdefined as one unit of TNF activity. Comparison with a preparation ofTNF-α (Genentech Inc., USA) showed that 1 unit in our assays was equalto 100-200 pg/ml TNF.

Cytokine neutralization assays.

The antisera used were: rabbit anti-human TNF-α, lot 2958-40 and rabbitanti-human IFN-γ, lot 2891-56 (Genentech Inc., USA), sheep anti-humanIFN-α,β, and sheep anti-human IFN-γ (obtained from Dr. K. Cantell,Finland). The cytokine preparations were treated with the sera diluted1:200 in culture medium and incubated for one hour at room temperature.Then, the residual IFN or TNF activities were determined as described.

Five different batches (L₁ to L₅) of PBL prepared from the blood ofhealthy blood donors were used. The optimal PBL concentration for theassays was found to be 8×10⁶ cells per one ml of medium (RPMI 1640supplemented with 10% fetal calf serum and antibiotics).

Incubation of human PBL with the new reaction products I-XI (Table 1)resulted in IFN and TNF synthesis. The observed responses were doserelated in the range of 3-100 μg/ml of the compounds (Table 2). Thecompounds used in the indicated concentrations were non-cytotoxic. Inall of the bioassays, negative and positive controls were included. Thenegative controls measured the amounts of the cytokines (IFN and TNF)produced spontaneously without the addition of any exogenous stimulants.The positive controls indicated the amounts of the cytokine produced inresponse to a known reference inducer; in our case this wasphytohemagglutinin (PHA, Pharmacia, Sweden, 10 μg per ml).

It should be pointed out that the cytokine induction in human PBLcultures obtained from different individuals usually gives considerablevariation of the results. The phenomenon may be explained in terms ofgenetic differentiation of human population. Furthermore, PBL culturesoften produce IFN and TNF spontaneously.

In other words, high responders and low responders or evennon-responders are commonly observed among the healthy donors of PBL.

Despite the presented limitations, the results of the bioassays showedthat PBL (L₁ to L₅) treated with the reaction products (I-XI) producedIFN and/or TNF that could be measured quantitatively.

In the case of L₁ which contained leukocytes of the high responder, thereaction product II (containing L-form of aspartic acid) was found to beconsiderably more active as a cytokine inducer than the reaction productIII (containing the D-form of aspartic acid which also is more expensiveby two orders of magnitude). The observation is significant becausemainly L-forms of the amino-acids are recognized by cells as naturalsubstrates in biochemical reactions.

Furthermore, for the expression of biological activity of the reactionproducts, the amino-acid part of the molecule is much more importantthan the form of sugar. Instead of single sugars,--preferably lowmolecular weight, especially of less than 1000 daltons--polysaccharides(such as dextranes, reaction products X-XI) can be used and they reactsimilarly.

And vise versa, polysaccharides containing the amino-acid residues mayhave biological activity, and this activity is retained when they aredecomposed to oligosaccharides with the bound amino-acid (data notshown).

Similar results may also be observed if a short peptide is taken insteadof a single amino-acid and is used to stimulate the leukocytes toproduce cytokines (data not shown).

Seven reference batches of the unfactionated TTP assayed in over 100 PBLcultures from different donors produced from 10 to 1,000 units of IFNper ml, and from 9 to 750 units of TNF per ml. The fraction EK₂ -Sprepared from a mixture corresponding to the contents of natural peatextract (Example 5) has been assayed in eight PBL cultures from eightdifferent blood donors. It was found to be the most active preparationin inducing both IFN and TNF (data not shown).

Possible applications of the reaction products are as immuno-modulatorsand such activity was clinically useful. Tissue regeneration is anotherproven activity. Anti-cancer activity probably connected with thepresence of the induced interferon and tumor necrosis factor was alsoobserved. Anti-viral activity was also noted.

The main use of the above reaction products involves oraladministration, but parenteral treatment is also possible, as is,topical application. The products appear relatively stable.

                  TABLE 1                                                         ______________________________________                                        List of Reaction products                                                     No.           Substrates                                                      ______________________________________                                        I (D-10)      L-glutamic acid, glucose, galactose                             II (D-11)     L-aspartic acid, glucose, galactose                             III (D-13)    D-aspartic acid, glucose, galactose                             IV (D-12)     L-serine, glucose, galactose                                    V             EK.sub.2 -S (fractions 11-13)                                   VI            EK.sub.2 -S (fractions 6-7)                                     VII           EK.sub.2 -S (fractions 8-10)                                    VIII          EK.sub.2 -S (fractions 28-34)                                   IX            L-proline, glucose                                              X             L-aspartic acid + dextrane (variety 1)                          XI            L-aspartic acid + dextrane (variety 2)                          ______________________________________                                    

EXAMPLE 12

Pharmaceutical formulations containing as an active ingredient thereaction products according to Examples 1 to 5, 9 and 10, were preparedusing the following reaction products:

A. Tablets/Granules:

5.0 g of the reaction product obtained according to Example 1 or 10(active substance),

444.0 g of pharmaceutically acceptable lactose

1.0 g of lubricant (e.g. MYVATEX®, trademark of Eastman Kodak)

The ingredients were mixed and granulated with 30% aqueous ethanol in aconventional way, then dried at 40° C. The granules were used to preparecapsules, each containing approx. 450 mg of granules, i.e. 5 mg of theactive substance. Alternatively, the granules were used to form tablets,each weighing approx. 450 mg and containing 5 mg of the activesubstance.

B. In the same conventional manner, the active substances obtainedaccording to the preceding examples 1 to 5, 9 and 10 were formulatedinto other pharmaceutical formulations using suitable carriers.

                                      TABLE 2                                     __________________________________________________________________________    Biological Activity of the Reaction Products I-XI in Human PBL                Dose    IFN Units/ml        INF units/ml                                      μg/ml                                                                              L.sub.1                                                                           L.sub.2                                                                           L   L   L   L   L   L   L   L                                 __________________________________________________________________________    Control                                                                           --  10  <10 <10 20  <10 80  9   27  27  <9                                PHA 10  100 30  30  60  100 250 80  250 250 250                               I   100 100 <10 <10 20  --  250 9   9   160 --                                    30  --  --  --  30  --  --  --  --  500 --                                    10  30  <10 <10 30  --  80  9   18  160 --                                    3   --  --  --  10  --  --  --  --  160 --                                II  100 100 10  <10 10  <10 250 18  18  250 <9                                    30  --  --  --  10  <10 --  --  --  250 <9                                    10  1000                                                                              10  30  10  10  250 50  27  250 <9                                    3   --  --  --  10  <10 --  --  --  160 <9                                III 100 <10 <10 10  10  <10 250 9   18  250 <9                                    30  --  --  --  30  <10 --  --  --  250 <9                                    10  <10 <10 <10 10  <10 80  27  18  250 <9                                    3   --  --  --  10  <10 --  --  --  80  <9                                IV  100 <10 10  10  20  <10 80  60  9   160 <9                                    30  --  --  --  20  <10 --  --  --  27  <9                                    10  <10 30  <10 20  <10 80  50  18  80  <9                                    3   --  --  --  20  <10 --  --  --  80  <9                                V   100 30  <10 <10 60  --  80  50  18  250 --                                    30  --  --  --  60  --  --  --  --  80  --                                    10  <10 <10 <10 10  --  80  27  9   80  --                                    3   --  --  --  10  --  --  --  --  80  --                                VI  100 <10 <10 <10 20  --  80  27  18  80  --                                    30  --  --  --  10  --  --  --  --  80  --                                    10  10  <10 <10 20  --  50  27  18  160 --                                    3   --  --  --  10  --  --  --  --  250 --                                VII 100 <10 <10 <10 --  --  80  27  27  --  --                                    30  --  --  --  --  --  --  --  --  --  --                                    10  10  <10 <10 --  --  80  27  27  --  --                                    3   --  --  --  --  --  --  --  --  --  --                                VIII                                                                              100 <10 <10 <10 --  --  80  160 27  --  --                                    30  --  --  --  --  --  --  --  --  --  --                                    10  <10 <10 <10 --  --  750 80  27  --  --                                    3   --  --  --  --  --  --  --  --  --  --                                IX  100 10  <10 <10 10  --  27  27  18  80  --                                    30  --  --  --  20  --  --  --  --  80  --                                    10  100 30  <10 20  --  80  27  18  80  --                                    3   --  --  --  10  --  --  --  --  50  --                                X   100 100 <10 20  20  --  27  27  18  250 --                                    30  --  --  --  30  --  --  --  --  80  --                                    10  <10 10  <10 30  --  18  50  27  50  --                                    3   --  --  --  30  --  --  --  --  250 --                                XI  100 <10 10  <10 30  --  18  27  27  50  --                                    30  --  --  --  10  --  --  --  --  250 --                                    10  <10 30  <10 10  --  18  80  80  80  --                                    3   --  --  --  20  --  --  --  --  750 --                                __________________________________________________________________________

EXAMPLE 13

The active substances obtained according to preceding Examples 1 to 5, 9and 10 were used as a beneficial additive to cosmetic preparationsintended for everyday hair and body care, the content of the substancesbeing within a range of 0.01-10% by weight, depending on the type of thepreparation, the method of application and the frequency of use intendedfor the particular preparation.

We claim:
 1. A composition comprising a mixture of at least two different Amadori rearrangement compounds of the general formula R₁ --NH--R₂, wherein R₁ is a 1-deoxy-2-ketose radical derived from a simple sugar, oligo- or polysaccharide, and R₂ is an amino acid or peptide radical, the mixture being capable of stimulating an immune system by inducing cytokine formation.
 2. The composition of claim 1, wherein at least one of the radicals is derived from a compound selected from the group consisting of an oligo- or polysaccharide having a molecular weight of 5000 daltons or less, and a peptide having a molecular weight of less than 1000 daltons.
 3. The composition of claim 1, wherein the mixture further comprises inorganic trace elements.
 4. The composition of claim 1, wherein the simple sugar is selected from the group consisting of glucose, xylose, galactose, rhamnose, fructose, mannose, 2-deoxy-glucose, 6-deoxy-glucose, glucosamine, and galactosamine.
 5. The composition of claim 1, wherein the amino acid radical is selected from the group consisting of serine, glycine, proline, histidine, arginine, alanine, aspartic acid, glutamic acid, phenylalanine, threonine, cysteine, cyrstine, glutamine, valine, asparagine, methionine, tyrosine, hydroxyproline, lysine, tryptophane, isoleucine, and leucine.
 6. The composition of claim 1, further comprising at least one substance selected from the group consisting of a pharmaceutically-acceptable carrier, a cosmetically acceptable carrier, an adjuvant, and a lubricant, a weight ratio of the at least one Amadori rearrangement compound to the at least one substance being between 1:1 to 1:100.
 7. The composition of claim 6, wherein the weight ratio ranges from 1:8 to 1:20.
 8. The composition of claim 6, wherein the weight ratio is about 1:9.
 9. The composition of claim 6, comprising a lubricant present in admixture with lactose, a weight ratio of lactose to the lubricant being between 20:1 and 100:1.
 10. The composition of claim 9, wherein the weight ratio of lactose to the lubricant is about 50:1.
 11. A cosmetic preparation comprising at least one cosmetically-acceptable carrier or additive and at least one Amadori rearrangement compound of the general formula R₁ --NH--R₂, wherein R₁ is a 1-deoxy-2-ketose radical derived from a simple sugar, oligo- or polysaccharide, and R₂ is an amino acid or peptide radical.
 12. The cosmetic preparation of claim 11, further comprising at least one other Amadori rearrangement compounds of the general formula R₁ --NH--R₂, wherein R₁ is a 1-deoxy-2-ketose radical derived from a simple sugar, oligo- or polysaccharide, and R₂ is an amino acid or peptide radical, the mixture being capable of stimulating an immune system by inducing cytokine formation.
 13. The cosmetic preparation of claim 12, wherein at least one of the radicals is derived from a compound selected from the group consisting of an oligo- or polysaccharide having a molecular weight of 5000 daltons or less, and a peptide having a molecular weight of less than 1000 daltons.
 14. The cosmetic preparation of claim 12, further comprising inorganic trace elements.
 15. The cosmetic preparation of claim 12, wherein the simple sugar is selected from the group consisting of glucose, xylose, galactose, rhamnose, fructose, mannose, 2-deoxy-glucose, 6-deoxy-glucose, glucosamine, and galactosamine.
 16. The cosmetic preparation of claim 12, wherein the amino acid radical is selected from the group consisting of serine, glycine, proline, histidine, arginine, alanine, aspartic acid, glutamic acid, phenylalanine, threonine, cysteine, cystine, glutamine, valine asparagine, methionine, tyrosine, hydroxyproline, lysine, tryptophane, isoleucine, and leucine.
 17. The cosmetic preparation of claim 12, further comprising at least one substance selected from the group consisting of a pharmaceutically-acceptable carrier, a cosmetically acceptable carrier, an adjuvant, and a lubricant, a weight ratio of the at least one Amadori rearrangement compound to the at least one substance being between 1:1 to 1:100.
 18. The cosmetic preparation of claim 17, wherein the weight ratio ranges from 1:8 to 1:20.
 19. The cosmetic preparation of claim 17, wherein the weight ratio is about 1:9.
 20. The cosmetic preparation of claim 17, comprising a lubricant present in admixture with lactose, a weight ratio of lactose to the lubricant being between 20:1 and 100:1.
 21. The cosmetic preparation of claim 20, wherein the weight ratio of lactose to the lubricant is about 50:1.
 22. The cosmetic preparation of claim 11, wherein the at least one Amadori rearrangement compound is present in an amount of 0.01-10% by weight.
 23. A cosmetic preparation according to claim 22, wherein the at least one Amadori rearrangement compound is represent in an amount of 0.01-1% by weight.
 24. A cosmetic preparation according to claim 22, wherein the at least one Amadori rearrangement compound is present in an amount of 0.05-0.10% by weight.
 25. A process for the manufacture of a mixture of at least two different Amadori rearrangement compounds as defined in claim 1, the process comprising the steps of:a) reacting at least one amino acid or peptide with at least one simple sugar, oligo- or polysaccharide in the presence of water as a solvent at a temperature of about 70°-121° C.; b) continuing said reaction to allow the resulting products to undergo Amadori rearrangement while simultaneously or subsequently removing the aqueous solvent, until the reaction mixture turns light orange brown in color and biological activity and ferricyanide reducing capacity can be detected in samples taken from the mixture; c) stopping the Amadori rearrangement before decomposition yields compounds that have lost their biological activity; and thereafter d) with drying the resulting mixture containing said at least two different Amadori rearrangement compounds or further subjecting that mixture to a purification by column chromatography and selectively collecting biologically active fractions that cause reduction of potassium ferricyanide.
 26. The process of claim 25, wherein said reaction is carried out for about 30 to 120 minutes.
 27. The process of claim 25, wherein the reaction is carried out in the presence of inorganic trace elements.
 28. The process of claim 27, wherein the reaction is carried out under pressure.
 29. The process of claim 27, wherein the reaction is carried out in the presence of a lower alcohol.
 30. The process of claim 28, wherein the reaction is carried out in the presence of a lower alcohol.
 31. The process of claim 25, wherein the molar ratio of sugars, oligo- or polysaccharides to the amino acids and peptides is between 2:1 and 1:1.
 32. The process of claim 25, wherein the mixture comprises at least one compound selected from the group consisting of an oligo- or polysaccharide having a molecular weight of 5000 daltons or less, and a peptide having a molecular weight of less than 1000 daltons.
 33. The process of claim 25, wherein at least one amino acid has two carboxyl groups and the reaction is carried out in the presence of a buffer salt in a molar ratio with the amino acid of 1:1.
 34. The process of claim 33, wherein the buffer salt is sodium bicarbonate.
 35. The process of claim 25 wherein the reaction is carried out in a concentrated aqueous solution of about 0.67 to about 2.75 pbw of solids per 1 pbw of aqueous solvent.
 36. The process of claim 25, further comprising the step of adding at least one pharmaceutically or cosmetically acceptable carrier or additive to the mixture of the Amadori rearrangement compounds obtained by step (d).
 37. A method of cytokine induction by polyclonal activation of mammalian cells, the method comprising the step of administering to mammalian cells a preparation comprising a cytokine induction effective amount of at least one biologically active Amadori rearrangement compound of the general formula R₁ --NH--R₂, wherein R₁ is a 1-deoxy-2-ketose radical derived from a simple sugar, oligo- or polysaccharide and R₂ is an amino acid or peptide radical.
 38. The method of claim 37, wherein the preparation comprises at least two different Amadori rearrangement compounds.
 39. The method of claim 38, wherein at least one of the radicals is derived from a compound selected from the group consisting of an oligo- or polysaccharide having a molecular weight of 5000 daltons or less, and a peptide having a molecular weight of 1000 daltons or less.
 40. The method of claim 38, wherein the mixture further comprises inorganic trace elements.
 41. The method of claim 38, wherein the simple sugar is selected from the group consisting of glucose, xylose, galactose, rhamnose, fructose, mannose, 2-deoxy-glucose, 6-deoxy-glucose, glucosamine, and galactosamine.
 42. The method of claim 38, wherein the amino acid radical is selected from the group consisting of serine, glycine, proline, histidine, arginine, alanine, aspartic acid, glutamic acid, phenylalanine, threonine, cysteine, cystine, glutamine, valine, asparagine, methionine, tyrosine, hydroxyproline, lysine, tryptophane, isoleucine, and leucine.
 43. The method of claim 38, wherein the preparation further comprises at least one substance selected from the group consisting of a pharmaceutically-acceptable carrier, a cosmetically acceptable carrier, an adjuvant, and a lubricant, a weight ratio of the at least one Amadori rearrangement compound to the at least one substance being between 1:1 to 1:100.
 44. The method of claim 43, wherein the weight ratio ranges from 1:8 to 1:20.
 45. The method of claim 43, wherein the weight ratio is about 1:9.
 46. The method of claim 43, wherein the preparation comprises a lubricant present in admixture with lactose, a weight ratio of lactose to the lubricant being between 20:1 and 100:1.
 47. The method of claim 46, wherein the weight ratio of lactose to the lubricant is about 50:1.
 48. The method of claim 46, wherein the Amadori rearrangement compound is administered as a prophylactic or therapeutic treatment to a human or animal body in need of such treatment.
 49. The method of claim 39, wherein the at least one Amadori rearrangement compound is administered as a prophylactic or therapeutic treatment to a human or animal body in need of such treatment. 